Previously, a novobiocin-hypersensitive mutant of Escherichia coli K-12 carrying a cysE-pryE linked mutation, designated rfaD, which specifically affects the synthesis of the aldoheptose, L-glycero-D-mannoheptose, has been isolated and genetically characterized. The following facts have been established: (i) The truncated lipopolysaccharide (LPS) of the rfaD mutant contains D-glycero-D-mannoheptose rather than the normal L-glycero-D-mannoheptose. (ii) The rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase is required for the conversion of ADP-D-glycero-D-mannoheptose to ADP-L-glycero-D-mannoheptose. (iii) The nucleotide ADP-D-glycero-D-mannoheptose accumulates in the rfaD strains. These results provide support for the following biosynthetic reactions: (a) the conversion of D-glycero-D-mannoheptose-l-P and ATP to ADP-D-glycero-D-mannoheptose and pyrophosphate; (b) ADP-D-glycero-D-mannoheptose is converted to ADP-L-glycero-D-mannoheptose. These two reactions are catalyzed by the enzymes ADP-D-glycero-D-mannoheptose-synthetase and ADP-L-glycero-D-mannoheptose-6-epimerase, respectively. The rfaD gene has been cloned into pBR322, and the purification of the rfaD gene product is in progress. Recently, we have isolated a new mutant carrying a cysE-pyrE linked mutation, designated rfa2, which results in an increased permeability to hydrophobic agents and a heptose-less LPS structure. Further, we have demonstrated that the rfa2 mutation is genetically distinct from the rfaD locus and that the rfa2 phenotype is not abolished by plasmid carrying the wild type rfaD allele. Studies of the biochemical alteration(s) in the rfa2 mutant are now being conducted.